This invention relates to factors involved in the coagulation pathway, and more particularly to serpins modified to selectively modulate the coagulation pathway.
The regulation of blood clotting is an important process in animals with a developed cardiovascular system. Clotting is achieved through either an intrinsic or extrinsic pathway that involves a cascade of protein activations resulting in the conversion of soluble fibrinogen to insoluble fibrin.
The intrinsic pathway includes the following steps: (1) factor XII is activated; (2) activated factor XII activates factor XI; (3) activated factor XI activates factor IX; (4) activated factor IX, with interaction from activated factor VIII, activates factor X; (5) activated factor X converts prothrombin to thrombin in the presence of activated factor V; (6) thrombin cleaves fibrinogen to fibrin; (7) fibrin polymerizes to form fibrin strands.
The extrinsic pathway includes the following steps: (1) trauma to the vessel wall causes binding of factor VII in plasma to tissue factor present in non-vascular tissue cells; (2) factor VII is activated; (3) factor VII-tissue factor complex activates Factor X. The remaining steps are the same as steps 5-7 of the intrinsic pathway.
A fine line must be maintained between the activation and inactivation of coagulation (Gaffney et al., 1999). If clotting proteins are constantly inactivated, then blood loses the ability to clot which could lead to life-threatening bleeding events.
Hemophilia A is an inherited factor VIII deficiency that results in extensive bleeding after trauma and may involve spontaneous bleeding into joints and muscles. In normal individuals, factor VIII circulates in the plasma bound to von Willebrand factor. Patients with Hemophilia A exhibit reduced levels of factor VIII, which results in a reduction in blood clotting. Patients with a level of factor VIII less than 1% of normal have severe bleeding episodes throughout life. A level of about 5% of normal results in few, if any, spontaneous bleeding episodes, but severe bleeding can occur in these patients, for example, following surgery, if not properly managed. Patients with factor VIII levels of about 10 to 30% of normal have very mild hemophilia, but may still experience excessive bleeding, for example, following surgery.
Treatment of patients with hemophilia A generally involves administering factor VIII. The factor VIII may be obtained from human donors, or from animals, for example, porcine factor VIII. Recombinantly produced factor VIII may also be used. Severe hemophiliacs require frequent infusions of factor VIII to restore the blood""s normal clotting ability. However, supplies of human factor VIII are often inadequate, and the time and expense involved in its isolation and purification from blood are considerable, especially in light of the risk of transmitting viruses such as AIDS and hepatitis.
About 15% of hemophilia A patients develop antibodies to factor VIII. These antibodies inhibit the anticoagulant activity of therapeutically administered factor VIII. Immune tolerance may be achieved through continuous exposure to factor VIII. This requires large and continuous infusions of factor VIII, which are costly, and, in the case of human-derived factor VIII, pose the risk of viral infection. Providing a means for extending the bioavailability of factor VIII would reduce the amount of factor VIII needed to treat hemophilia A patients.
In the opposite situation, if clotting factors are not degraded or inhibited once clotting begins, the blood clot can quickly spread within vessels and capillaries blocking blood flow to vital organs. Some of the leading causes of death in this country are the result of clotting diseases like stroke and myocardial infarction. Regulation of blood clotting will greatly influence therapeutic control over clotting and clotting diseases.
The steps involved in forming a clot include a series of zymogen activations (Roberts and Lozier, 1992). Zymogens are precursors of enzymes that are activated once they are proteolytically cleaved. The cleavage of the zymogen alters the protein structure exposing an active site and allowing enzymatic activity to occur. The coagulation process is interesting because many of the zymogens, when cleaved, become active serine proteases which have a specificity to cleave the next zymogen in the reaction. The activation of successive serine proteases provides a rapid response to a relatively small signal. Several steps in the process can be regulated to activate or inhibit clot formation.
Within the coagulation cascade, many of the serine proteases can also cleave inhibitors of the clotting process. The inhibitors are members of the family of proteins known as serpins (serine protease inhibitors) (Potempa et al., 1994). Often, when a serpin is cleaved by a serine protease, the two proteins remain covalently bound, which inhibits the protease by preventing it from reacting with any other molecules (Mammen, 1998). One of the best examples of this is the interaction between the serine protease thrombin and the serpin antithrombin (AT).
Thrombin is the final protease generated in the clotting cascade, and is activated by the cleavage of prothrombin by Factor Xa (FIG. 1A). Thrombin can then cleave fibrinogen into fibrin monomers. Polymerization of fibrin monomers forms the fibrin part of the clot. Though activating fibrin is the main function of thrombin, it also has other functions. Thrombin can activate a positive feedback pathway by proteolytically activating Factors V and VIII that assist in the activation of prothrombin into thrombin. Thrombin also proteolytically cleaves proteins that can serve as inhibitors of its action.
Antithrombin is the most important anticoagulant in the blood (FIG. 1B). It has a high specificity for thrombin, but also weakly inhibits several other serine proteases. Antithrombin inhibits thrombin by inserting its reactive site loop into the active site of thrombin. The interaction forms a stable complex between the two molecules (Mammen, 1998).
The protease activity of thrombin cleaves the reactive site loop of antithrombin between an arginine and serine which are labeled P1 and P1xe2x80x2 for the site of thrombin cleavage. The cleavage results in a covalent bond between antithrombin and thrombin and prevents thrombin from carrying out any further proteolytic reactions (Olson et al., 1995).
The binding between AT and thrombin occurs very slowly (k2=9xc3x97103 Mxe2x88x921sxe2x88x921) (Olson and Shore, 1982). In its native form, the reactive site loop of AT is not easily accessible to thrombin (Jin et al., 1997). Heparin, a polysaccharide with a strong negative charge, helps to speed this reaction (Olson and Shore, 1982). A specific pentasaccharide in heparin binds to the positively charged D-helix of AT (Jin et al., 1997; Olson et al., 1992). This causes AT to go through a structural change allowing the reactive site loop on AT to be more accessible to thrombin (Ersdal-Badju et al., 1997; Huntington and Gettins, 1998; Meagher et al., 1996). Heparin also binds to thrombin and acts as a bridge to draw the two molecules together (Danielsson et al., 1986). When heparin is bound to AT, the rate of reaction with thrombin is increased significantly (500- to 1000-fold) (Olson and Shore, 1982).
Although thrombin usually functions as a procoagulant, it can also activate the protein C pathway, an anticoagulant pathway. Thrombin has the ability to bind to an endothelial cell receptor called thrombomodulin (TM). When thrombin is bound to TM, it goes through a conformational change that results in a change in substrate specificity for protein C instead of fibrinogen (FIG. 1A) (Ye et al., 1991). Protein C interacts with thrombin through Ca2+ bridges (Rezaie and Esmon, 1992). The protein C is cleaved by the thrombin bound to TM, generating activated protein C (APC). APC proteolytically degrades two cofactors of clotting, Factor Va and Factor VIIIa, preventing the activation of prothrombin to thrombin. Once activated, APC activity is accelerated when it is complexed with the cofactors protein S and Factor V. In other words, thrombin bound by TM becomes an anticoagulant by activating APC and forming a negative feedback pathway for thrombin activation.
Another member of the serpin family is protein C inhibitor (PCI). It has been found to serve several vital roles in the regulation of coagulation (FIG. 1B). Protein C inhibitor is a unique serpin because it has the ability to work as a procoagulant and an anticoagulant. Protein C inhibitor inhibits APC both directly and indirectly (Neese et al., 1994). It directly blocks APC activity by binding to its active site, similar to inhibition of thrombin by AT. When APC is inhibited by PCI, APC cannot degrade the clotting factors that activate prothrombin, Factors Va and VIIIa. Protein C inhibitor also has an increased ability to inhibit thrombin bound to TM (Elisen et al., 1998; Rezaie et al., 1995). When TM-bound thrombin is inhibited by PCI, protein C cannot access it, which prevents activation of protein C. The inhibition of APC and TM-bound thrombin by PCI blocks both the activation and activity of protein C allowing the production of thrombin to continue. By inhibiting the negative feedback pathway, PCI acts as a procoagulant.
Protein C inhibitor also can directly inhibit thrombin. The reactive site loop of PCI is compatible with the active site of thrombin and when cleaved it forms a covalent bond similar to that between AT and thrombin (Cooper and Church, 1995). Based on inactivation reactions in the absence of heparin, PCI inhibits thrombin faster than AT suggesting that its reactive site loop fits better in the active site of thrombin (Rezaie, 1997). While heparin increases the activity of AT with thrombin by 300-fold, it only increases the activity of PCI with thrombin by 20- to 30-fold (Shirk et al., 1994). This difference suggests that heparin binding does not cause as many structural changes in PCI as in AT.
As mentioned previously, thrombin binding to TM does not prevent its inactivation by the serpins. Inhibition by AT increases when the thrombin is bound to TM, but only if a heparin-like polysaccharide, chondroitin sulfate, is also attached to the TM (Bourin et al., 1986; Bourin et al., 1988). It is believed that the chondroitin sulfate binds to the heparin binding site of AT and the heparin binding exosite 2 of thrombin and functions similarly to heparin (Weisel et al., 1996). However, the increase in activity is fairly small (4-fold) compared to free thrombin in the presence of heparin (300-fold) (Preissner et al., 1987). While the activity of PCI with free thrombin is fairly low (k2=1.7xc3x97104 Mxe2x88x921sxe2x88x921), when thrombin is bound by TM, PCI becomes a very strong inhibitor of thrombin activity (k2=2.4xc3x97106 Mxe2x88x921sxe2x88x921) (Rezaie et al., 1995). Though AT needs chondroitin sulfate to have a pronounced effect on TM-bound thrombin, PCI is not as sensitive to chondroitin sulfate (2-fold greater than without chondroitin sulfate) (Rezaie et al., 1995). Because heparin does not have a strong affect on PCI, it could be assumed that chondroitin sulfate would not affect PCI as strongly either.
The most important domains of TM are a series of six epidermal growth factor (EGF)-like motifs. It has been determined that domains 5 and 6 are responsible for TM binding thrombin, but thrombin cannot activate protein C when it is bound by these two regions alone (Ye et al., 1991). When domain 4 is included with 5 and 6, thrombin changes its conformation giving it similar enzymatic rates to thrombin complexed with full membrane-bound TM (Hayashi et al., 1990).
Although the general structures and functions of PCI and AT are similar, different amino acid sequences in the heparin binding domains and reactive site loops are believed to be responsible for the observed differences in function (Cooper and Church, 1995). Molecular modeling and mutagenesis studies have shown that heparin binding sites differ between AT and PCI (Cooper et al., 1995; Neese et al., 1998). In AT, heparin binds to the D-helix region (Jin et al., 1997), but in PCI, it has been found to bind to the H-helix (Neese et al., 1998). The H-helix on both proteins is in a position that would require heparin to bend in order to bind both thrombin and the serpin. This may be a reason why heparin and chondroitin sulfate have a reduced affect on acceleration of thrombin inhibition by PCI compared with AT.
There are important differences between the sequence of the H-helix of AT and PCI, as shown below in Table 1. The difference in the sequence of the H-helix could affect how each protein interacts with heparin and inhibits thrombin. Heparin binds to proteins using positively charged residues on xcex1-helices. The H-helix of AT contains amino acid residues with negative charges, but the H-helix of PCI is mainly composed of positive and neutral residues (Shirk et al., 1994). First, two positive residues on the H-helix of PCI, Arg269 and Lys270, are known to contribute to heparin interactions with PCI (Shirk et al., 1994). The corresponding positions of AT contain a neutral Gln305 and a negative Glu306 which would repel heparin (Shirk et al., 1994). Second, the carboxy terminus of the H-helix is also considerably different between the two serpins. Protein C inhibitor contains positive and neutral residues while AT has mainly negative residues (Shirk et al., 1994). It is unknown whether this region of the helix is important for PCI to bind heparin. The negative charges in this region in AT could contribute steric effects and prevent heparin from binding to the H-helix on AT. Third, another difference near the H-helix is the presence of an arginine at position 278 in PCI that may form a salt bridge with Glu39 of thrombin in complexes with PCI, but AT has a methionine in the corresponding position at residue 314 (Cooper and Church, 1995).
Another region in which differences in sequence could greatly affect thrombin inhibition is in the reactive site loop. Differences in inhibition rates among serpins suggest that reactive site loops may possess residues that fit better with different conformations of thrombin. For example, PCI inhibits thrombin bound to TM better than free thrombin suggesting that a conformational change in the active site of thrombin allows it to accommodate the residues in the reactive site loop of PCI (Le Bonniec and Esmon, 1991; Rezaie, 1997; Rezaie et al., 1995).
Comparison of the sequences of the reactive site loops of AT and PCI (shown below in Table 2) demonstrates that between P3 and P3xe2x80x2, only the P1 and P1xe2x80x2 residues are conserved. The P1 arginine and P1xe2x80x2 serine are important to proper serpin function because thrombin cleaves the reactive site loop between these two residues (Stephens et al., 1988). While the surrounding residues are not directly involved in the cleavage, they are inserted into the active site of thrombin and contribute to the association between the reactive site loop and the active site loop of thrombin. Because thrombin can be found in several different conformations, mutations in the reactive site loop of the serpins would be useful to modulate the insertion of the reactive site loop into different configurations of the active site of thrombin, thereby modulating thrombin activity and consequently modulating coagulation. This would be especially useful in the treatment of diseases involving deficiencies of coagulation factors.
In summary, agents for modulation of the clotting cascade, for example that modulate thrombin activity, are needed for the treatment of clotting diseases.
The invention provides mutants of antithrombin (AT) modified in the H-helix and reactive site loop sequence, having modulated activity in the inhibition of thrombin. In particular, AT modified to have a greater positive charge in the H-helix (AT-pos), has enhanced inhibitory activity against thrombin bound to thrombomodulin (T-TM), at a level which resembles the procoagulant activity of Protein C Inhibitor (PCI). The AT-pos mutants retain AT ability to inhibit free thrombin, but, due to its enhanced activity against T-TM, the AT-pos mutants effectively shut down the T-TM activation of Protein C, inhibiting Activated Protein C degradation of Coagulation Factors V, VIII, and X.
The invention provides nucleic acids encoding AT-pos mutants, amino acid sequences of AT-pos mutants, pharmaceutical compositions containing AT-pos mutants, and methods of treating patients deficient in one or more coagulation Factors, for example, hemophiliacs, and methods for extending the bioavailability of Factor VIII in a patient. In particular, the invention provides AT-pos mutants and methods for their use in extending the bioavailability of Factor VIII.